Biosynthetic thiolases catalyse carbon-carbon bond formation via a thioester-dependent Claisen-condensation reaction mechanism. This is an essential first step of many biosynthetic pathways relying on the stepwise assembly of carbon backbones from 2- and 3-carbon metabolites, including fatty acids and lipids, polyketides, isoprenoids, cholesterol, steroid hormones and ketone bodies. Many of these compounds are industrially important chemicals due to their biological activity or potential application as building blocks or substrates for the production of bulk chemicals and pharmaceuticals (Klein, M. (2009) Enzyme and Microbial Technology, 45, p. 361-366).
Biosynthetic thiolases (EC 2.3.1.9) such as BktB from Ralstonia eutropha are also involved in poly-hydroxalkanoate biosynthesis, and serve to condense either two acetyl-CoA's to form acetoacetyl-CoA in polyhydroxybutyrate (PHB) biosynthesis or to condense acetyl-CoA with propionyl-CoA or butyryl-CoA to form valeryl-CoA or hexanoyl-CoA (Kim, Eun-Jung et al., 2014, Biochemical and Biophysical Research Communications, 444, 3, p. 365-369).
Sustainable production of advanced biofuels and chemicals from renewable feedstocks requires metabolic engineering of microorganisms to synthesise longer carbon chain length compounds from 2- and 3-carbon metabolic intermediates. Invariably, this requires C—C-bond formation by biosynthetic thiolases. For example, reversal of the β-oxidation cycle provides a platform for the synthesis of fatty acids and fatty acid-derived chemicals, using biosynthetic thiolases to initiate and reverse the cycle so that the carbon backbone is extended rather than degraded (Clomburg, James et al., 2015, Metabolic Engineering 28, p. 202-212). This reverse β-oxidation cycle can be used to generate a diverse range of products (Cintolesi, Angela et al., 2014, Metabolic engineering 23, p. 100-115; Dellomonaco, 2011, Nature, vol 476, p. 355-359). The intermediates of the reverse β-oxidation cycle can be removed from the cycle to form 3-keto-fatty acids or the corresponding methyl ketones via decarboxylation, medium chain length polyesters, fatty aldehydes, fatty alcohols, fatty acids, alkanes, and alkenes (Yu, Ai-Qun, 2014, Frontiers in Bioengineering and Biotechnology, Vol 2, article 78).
The products from the reverse β-oxidation cycle can also be transferred from the reverse β-oxidation cycle to the fatty acid biosynthesis (FAS) cycle by employing enzymes that transfer the acyl-phosphopantetheine group from acyl-CoA to apo-ACP such as sfp-type PPTase (phosphopantetheinyl transferases) (Methods in Enzymology, Volume 458, Chapter 10; Beld, Joris et al., 2014, Nat. Prod. Rep. 31, 61-108). The fatty-acyl-ACP's can similarly be removed from the FAS cycle by a variety of enzymes to produce fatty acids and fatty-acid derived chemicals such as hydroxy fatty acids, fatty aldehydes, fatty alcohols, alkenes, and dicarboxylic acids (Janβen & Steinbüchel, 2014, Biotechnology for Biofuels, 7:7).
Supplying 3-keto-acyl-ACP substrates produced by biosynthetic thiolases followed by transfer of the acyl-phosphopantetheine group to apo-ACP, also provides an alternative entry of 3-keto-acyl-ACP's into the FAS cycle not relying on KAS III type β-ketoacyl-ACP synthases (EC 2.3.1.180, FabH) that require malonyl-ACP as extender to improve the overall carbon and energy efficiency of product synthesis (Dellomonaco, 2011, Nature, vol 476, p. 355-359).
Enzymes that are capable of C—C bond formation to condense acetyl-CoA with acids or CoA activated acids to form 3-keto-acyl-CoA esters, such as biosynthetic thiolases, are thus essential enzymes, not only in the synthesis of fatty acids and fatty acid-derived chemicals by providing the 3-keto-acyl-CoA or 3-keto-acyl-ACP intermediates to either the reverse β-oxidation or the FAS cycle, but also in polyhydroxyalkanoate biosynthesis and in the production of fermentation products such as butanol, butyric acid, acetone and hydrogen by clostridia (Klein, M. 2009. Enzyme and Microbial Technology, 45, 361-366), and many other biochemical pathways such as isoprenoids and polyketides.
However, all known biosynthetic thiolases have certain limitations. For example, they require two cysteine residues for their catalytic mechanism. In the acyl transfer step, Cys 378 protonates the CoA leaving group, and the acetyl group is transferred to Cys 89. In the subsequent Claisen condensation reaction, the deprotonated Cys378 abstracts the proton of the C2 atom of acetyl-CoA, facilitating its nucleophilic attack on the carbonyl carbon of the acetyl group that is covalently bound to the Cys 89 sulfur atom, which leads to C—C bond formation and release of the acyl group from Cys 89. This two-step “ping-pong” mechanism is also found in the biosynthetic thiolases involved in PHA biosynthesis of haloarchae, but in this case, Cys 89 is replaced by a Ser, leading to a Ser-His-Cys catalytic triad rather than a Cys-His-Cys triad found in other thiolases (Hou, Jing et al., 2013, Applied and Environmental Microbiology, Vol 79, number 17, p 5104-5111). Substrates with electrophilic groups, such as acrylic acid thioesters and haloacetyl-CoA analogs, irreversibly inactivate biosynthetic thiolases through both acylation of Cys 89 and alkylation of Cys 378 (Palmer, M. A. et al., J Biol Chem, 264 (1991), pp. 15293-15297; Palmer, M. A. et al., J Biol Chem, 266 (1991), pp. 8369-8375; Davis, Jeffrey T. et al., J Biol Chem, 262 (1987) pp. 90-96). For further example, biosynthetic thiolases are restricted to short chain substrates (C4 or shorter) such as acetyl-CoA, propanoyl-CoA, and butanoyl-CoA. Therefore, the longest acyl chain accepted by biosynthetic thiolases consist of only 4 carbon atoms due to the shape of the substrate binding pocket (Modis, Yorgo and Wiernga, Rik K. 1999, Structure, Vol 7 no. 10 p. 1279-1290).